Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera by a SPR assay. The glycopeptide CSF114(Glc) has been immobilized on a gold sensor chip and used for the screening of healthy blood donors’ inhibitor Tofacitinib and MS patients’ serum samples. SPR-assay data are compared with the previously validated ELISA method.2.?Experimental Section2.1. ReagentsGlycopeptide antigen CSF114(Glc) was prepared by microwave-assisted solid phase peptide synthesis. The glycopeptide was purified to homogeneity by solid phase extraction and reverse phase high-pressure liquid chromatography (HPLC), and further characterized by mass spectrometry and analytical HPLC as described elsewhere [37].Sensor chip CM5 and the running buffer HBS-EP+ 10�� (0.1 M HEPES, 1.
5 M NaCl, 30 mM EDTA and 0.5% v/v Surfactant P20; yielded pH 7.4 when diluted) were purchased from Biacore AB (GE Healthcare, Uppsala, Sweden). The amine coupling reagents N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 1 M ethanolamine hydrochloride-NaOH pH 8.5 were provided by Biacore Inhibitors,Modulators,Libraries AB (GE Healthcare). Sodium acetate was purchased from Carlo Erba (Milano, Italy). Sodium hydroxide was provided by Honeywell-Riedel deHaen (Seelze, Germany). All analyses were performed in a Biacore T100 instrument (GE Healthcare). All experiments were made at Inhibitors,Modulators,Libraries 25 ��C using HBS-EP+ as running buffer.2.2. Serum CollectionOne hundred and twenty one human serum samples were obtained for diagnostic purposes from patients and healthy blood donors who had given their informed consent.
Each serum sample was Inhibitors,Modulators,Libraries centrifuged and supernatant aliquoted and stored at ?20 ��C until use. Patients’ sera were obtained from a Inhibitors,Modulators,Libraries group of 61 relapsing��remitting MS (RR-MS) patients after a diagnostic lumbar puncture. Cerebrospinal fluid analysis and MRI examinations were performed for diagnostic purposes.2.3. Surface Plasmon Resonance (SPR)A stock solution of glycopeptide CSF114(Glc) was prepared in pure water (1 ��g/��L) and stored at +4 ��C. Immediately prior to immobilization procedure, peptide stock solution was diluted to a concentration of 10 ��g/mL in 0.1 mM sodium acetate pH 5.5. Standard amine coupling procedure was employed for Drug_discovery glycopeptide immobilization, essentially according to the Biacore procedures.
chemical information The appropriate flow cell of the sensor chip surface was activated by injecting an EDC/NHS (50:50) mixture at a flow rate 10 ��L/min during 420 s. CSF114(Glc) was injected at 10 ��L/min using the aim of immobilization procedure to give a final immobilization level of 800 �� 100 Resonance Units (RU). Unreacted groups on sensor chip surface were blocked by injecting 60 s-pulses of 1 M ethanolamine at pH 8.5 until complete deactivation. Reference channel was activated and subsequently blocked with ethanolamine.